Evaluation of Proenkephalin A 119–159 for liberation from renal replacement therapy: an external, multicenter pilot study in critically ill patients with acute kidney injury

Introduction Recent evidence suggests an association of plasma Proenkephalin A 119–159 (penKid) with early and successful liberation from continuous renal replacement therapy (CRRT) in critically ill patients with acute kidney injury. However, these exploratory results are derived from a monocentric trial and therefore require external validation in a multicenter cohort. Methods Data and plasma samples from the “Effect of Regional Citrate Anticoagulation versus Systemic Heparin Anticoagulation During Continuous Kidney Replacement Therapy on Dialysis Filter Life Span and Mortality Among Critically Ill Patients With Acute Kidney Injury—A Randomized Clinical Trial” (RICH Trial) were used for this validation study. PenKid was measured in all plasma samples available at CRRT initiation and at day 3 of CRRT. Patients were categorized into low and high penKid groups with a cutoff at 100 pmol/l. Competing-risk time-to-event analyses were performed. Competing risk endpoints were successful and unsuccessful liberation from CRRT, the latter meaning death or initiation of a new RRT within one week of discontinuation of primary CRRT. Then penKid was compared to urinary output. Results Low pre-CRRT penKid levels at CRRT initiation were not associated with early and successful liberation from CRRT compared to patients with high pre-CRRT penKid levels [subdistribution hazard ratio (sHR) 1.01, 95% CI 0.73–1.40, p = 0.945]. However, the landmark analysis on day 3 of ongoing CRRT demonstrated an association between low penKid levels and successful liberation from CRRT (sHR 2.35, 95% CI 1.45–3.81, p < 0.001) and an association between high penKid levels and unsuccessful liberation (sHR 0.46, 95% CI 0.26–0.80, p = 0.007). High daily urinary output (> 436 ml/d) was even stronger associated with successful liberation (sHR 2.91, 95% CI 1.80–4.73, p < 0.001) compared to penKid. Discussion This study suggests that penKid may be a competent biomarker to monitor the recovery of kidney function during CRRT. This is in line with previous findings and investigated this concept in a multicenter cohort. Again, low penKid was associated with early and successful CRRT liberation, but was outperformed by high daily urinary output. The findings of this study now warrant further evaluation in prospective studies or a randomized controlled trial. Trial registration The RICH Trial was registered at clinicaltrials.gov: NCT02669589. Registered 01 February 2016. Supplementary Information The online version contains supplementary material available at 10.1186/s13054-023-04556-w.

: Estimated cumulative incidence functions with log-log transformed pointwise 95% confidence intervals of (a), (c) successful liberation from CRRT and (b), (d) unsuccessful liberation from CRRT for the groups based on penKid value (≤ 89 pmol/l vs. > 89 pmol/l) before CRRT and based on the value at day 3 (landmark). The required relapse-free period after CRRT liberation to be classified as successfully liberated was set to 7 days.    Figure S4: Estimated cumulative incidence functions with log-log transformed pointwise 95% confidence intervals of (a), (c) successful liberation from CRRT and (b), (d) unsuccessful liberation from CRRT for the groups based on daily urinary output (> 436 ml/d vs. ≤ 436 ml/d) before CRRT and based on the value at day 3 (landmark). Urinary output on day 0 was recorded in 364/369 patients. Urinary output on day 3 was measured in all 150 landmark patients. The required relapse-free period after CRRT liberation to be classified as successfully liberated was set to 7 days.

Figure S5: Cumulative incidences separated by randomization group (heparin versus citrate anticoagulation) at baseline (pre-CRRT) and landmark time point (relapse-free period 7 days)
Time in days from start of CRRT  Figure S5: Estimated cumulative incidence functions with log-log transformed pointwise 95% confidence intervals of (a), (c) successful liberation from CRRT and (b), (d) unsuccessful liberation from CRRT for the randomization groups of the RICH trial at baseline (pre-CRRT) and landmark time point. The required relapse-free period after CRRT liberation to be classified as successfully liberated was set to 7 days.

Figure S6: Cumulative incidences separated by penKid group at baseline (pre-CRRT) and landmark time point (cut-off 100 pmol/l, relapse-free period 2 days)
Time in days from start of CRRT  Figure S6: Estimated cumulative incidence functions with log-log transformed pointwise 95% confidence intervals of (a), (c) successful liberation from CRRT and (b), (d) unsuccessful liberation from CRRT for the groups based on penKid value (≤ 100 pmol/l vs. > 100 pmol/l) before CRRT and based on the value at day 3 (landmark). The required relapse-free period after CRRT liberation to be classified as successfully liberated was set to 2 days.  Figure S7: Estimated cumulative incidence functions with log-log transformed pointwise 95% confidence intervals of (a), (c) successful liberation from CRRT and (b), (d) unsuccessful liberation from CRRT for the groups based on penKid value (≤ 100 pmol/l vs. > 100 pmol/l) before CRRT and based on the value at day 3 (landmark). The required relapse-free period after CRRT liberation to be classified as successfully liberated was set to 90 days.

Figure S8: Cumulative incidences separated by urinary output group at baseline (pre-CRRT) and landmark time point (cut-off 436 ml/d, relapse-free period 2 days)
Time in days from start of CRRT  Figure S8: Estimated cumulative incidence functions with log-log transformed pointwise 95% confidence intervals of (a), (c) successful liberation from CRRT and (b), (d) unsuccessful liberation from CRRT for the groups based on daily urinary output (> 436 ml/d vs. ≤ 436 ml/d) before CRRT and based on the value at day 3 (landmark). Urinary output on day 0 was recorded in 364/369 patients. Urinary output on day 3 was measured in all 150 landmark patients. The required relapse-free period after CRRT liberation to be classified as successfully liberated was set to 2 days.

Figure S9: Cumulative incidences separated by urinary output group at baseline (pre-CRRT) and landmark time point (cut-off 436 ml/d, relapse-free period 90 days)
Time in days from start of CRRT  Figure S9: Estimated cumulative incidence functions with log-log transformed pointwise 95% confidence intervals of (a), (c) successful liberation from CRRT and (b), (d) unsuccessful liberation from CRRT for the groups based on daily urinary output (> 436 ml/d vs. ≤ 436 ml/d) before CRRT and based on the value at day 3 (landmark). Urinary output on day 0 was recorded in 364/369 patients. Urinary output on day 3 was measured in all 150 landmark patients. The required relapse-free period after CRRT liberation to be classified as successfully liberated was set to 90 days. Missing 20 (5.4%) 9 (8.6%) 11 (4.2%)

APACHE II
Median ( Table S1: Patients characteristics at enrollment; all variables were collected at the time of randomization except for estimated GFR and creatinine, which were measured at the start time of CRRT. a Fisher's exact test comparing the low and high pre-CRRT penKid group. b Mann-Whitney U test comparing the low and high pre-CRRT penKid group. Abbreviations: APACHE II, Acute Physiology and Chronic Health Evaluation II, GFR, Glomerular Filtration Rate; SD, Standard Deviation; SOFA, Sequential Organ Failure Assessment

Statistical analysis
Statistical analyses were conducted using R (Version R-4.1.2 for Windows) i and the publicly available packages ComparisonCR, dplyr, haven, pROC, prodlim, rstatix, survival, survminer, and table1 were used. All analyses were conducted as exploratory analyses of hypothesis generation and were therefore not adjusted for multiple testing. All p-values and confidence limits were two-sided and were intended to be exploratory, not confirmatory. In this exploratory sense, p-values ≤0.05 were considered as statistically significant.
Baseline variables were assessed and as applicable, frequencies, percentages, medians, quartiles, means, standard deviations, and p-values were calculated. To compare baseline characteristics between pre-CRRT penKid groups (low vs. high), Fisher's exact test was used to compare categorical variables. Continuous variables were compared using the Mann-Whitney U test. Group characteristics were evaluated in the same way at the time of landmark analysis (day 3 of CRRT).
To answer our research question, we investigated the association of the pre-CRRT penKid group (penKid ≤ 100 pmol/l vs. penKid > 100 pmol/l) with the time to liberation from CRRT considering two competing events: successful and unsuccessful liberation from CRRT. Patients who survived and did not receive any form of RRT for at least 7 days (relapse-free period) after CRRT discontinuation were classified as successfully liberated. Patients who died within 7 days after CRRT discontinuation or who started a new RRT during this period were classified as unsuccessfully liberated. Patients who were not followed up for at least 7 days after discontinuation of CRRT were censored at the end of CRRT. Patients who required CRRT after day 28 were censored at day 28.
For both competing outcomes, we estimated the cumulative incidence (CIF) using the Aalen-Johansen estimator ii . Gray's k-sample test was applied to compare the cumulative incidence of the corresponding event type between penKid groups iii . Comparisons of the cumulative incidence functions at fixed time points were performed with the methods proposed by Chen at al. using Gaynor's variance and log-log transformed cumulative incidence functions iv . To quantify the association between the penKid group and the incidence of each competing event, we fitted two univariate Fine and Gray models, resulting in an estimate of the subdistribution hazard ratio (sHR) for each competing event v .
To investigate the predictive power of penKid also in patients during ongoing CRRT, we performed a landmark analysis on day 3 of CRRT. For this purpose, we selected all patients who were still at risk (i.e. receiving CRRT) on day 3 of CRRT and defined their penKid measurement on day 3 as the landmark penKid value. We then repeated the statistical analyses described above with this landmark penKid value for the time interval from day 3 to day 28, again dividing the patients into two groups with low and high landmark penKid values, using the same cut-off value of 100 pmol/l as before. Event times were thus recalculated starting from the landmark time point. In order to compare both analyses (pre-CRRT and landmark) visually, the same time axis was used in the figures.
The choice of the penKid cut-off value was partially justified by a sensitivity analysis of the landmark analysis. To do so, we fitted Fine & Gray models for "successful liberation from CRRT" of the above type for each possible and clinically meaningful penKid cutoff value present in the data and plotted the associated sHR and p-value against the cut-off. We repeated the sensitivity analysis for the pre-CRRT penKid analysis to test the robustness of the results.
To compare the performance of penKid to that of daily urinary output (> 436 ml/d vs. ≤ 436 ml/d) and to investigate whether the randomization group (Systemic Heparin Anticoagulation vs Regional Citrate Anticoagulation) might also affect the competing outcomes, we estimated CIFs and fitted univariate Fine & Gray models in the same manner as for penKid. In search of a suitable cut-off for daily urinary output, we repeated the sensitivity analysis as described above. To examine whether our results were robust to changes in the relapse-free period chosen in the outcome definition, we repeated the main analyses with a relapse-free period of 2 and 90 days.
Finally, we fitted a multivariable Fine & Gray model with penKid, age and diabetes predicting "successful liberation from CRRT" at landmark analysis to adjust our main result on potentially confounding baseline characteristics. These were selected by statistically significant differences between penKid groups at landmark.